Dimension of Time: From Culture to Assay

Our new cell-by-cell analysis module allows users to ‘count and classify’ heterogeneity in cell populations over time.

This software uses novel algorithms to segment the HD phase-contrast images which facilitates (1) label-free true cell counting (2) extraction of basic morphological features of individual cells (e.g. size, shape), and (3) measurement of fluorescence intensity that originates from within each cell (‘fluorescence within a phase boundary’). The ability to analyse at a cell-by-cell level will enable the characterisation of subsets and the power to monitor these individual groups in their response to various stimuli.

In this webinar Clare discusses:

• Overview and validation of the cell-by-cell analysis module
• How cell-by-cell analysis enables monitoring and quantification of dynamic phenotypic changes in heterogeneous populations
• Examples of how the software may be used for cell subset identification in order to provide an end-to-end solution for the study of heterogeneous populations in 96 well format

The last decade has seen a significant shift in the way that mammalian cells are used in biomedical science. Researchers are increasingly turning from simple, reductionist recombinant cells to primary cells, stem-cell derived cells and advanced in vitro co-culture and organoid models for greater translation and wider application in their work. However, developing and using these different cell systems presents fresh challenges to the everyday researcher. These relate to workflows for cell handling, genetic manipulation, assay development, phenotypic analysis and clinical use in the case of cell therapy.

• Changing landscape of cellular research
• Overview of new requirements to monitor these advanced cell systems
• How continuous live-cell imaging and analysis are aligned to the new biological models

Biomedical researchers are increasingly using advanced human cell models for translational biology and therapeutic discovery studies. These include patient-specific samples, cultured micro-tissues, and complex systems assembled from primary and iPS-derived cells. In line with this, information-rich and turnkey analytical techniques are demanded to extract maximum insight from these precious samples. Accordingly, non-invasive automated live-cell analysis methods have now become an industry gold-standard.

In this webinar, Del Trezise will review live-cell analysis techniques and the most recent developments in the field. Advances will be illustrated with examples drawn from 3D oncology co-culture applications, antibody-based cell identification methods, and cell-by-cell analysis to probe immune cell heterogeneity and subsets.

In this webinar you’ll learn:

• The basics of live-cell analysis and ‘smart cell monitoring’
• How live-cell analysis can be combined with advanced cell models for added insight and productivity
• What’s new in the field
• What advances researchers can expect in the future

Despite exciting recent developments in neuroscience, identifying novel, truly effective treatments for patients with neurological and psychiatric conditions remains highly challenging. There is high unmet need for more translational models and advanced analytical techniques that afford greater biological insight and productivity.

For cell-based assays, traditional approaches rely on end-point readouts (e.g. immunocytochemistry, high content imaging) and short term functional measurements (e.g. electrophysiology). These methods are not best suited to probing neuronal plasticity and the long term changes that occur during development, learning, and the inevitable decline in age and disease. 

In this webinar, Susana L. Alcantara will discuss novel live-cell analysis techniques and their applications in neuroscience. Data on a range of advanced cell models and phenotypic readouts that are amenable for neuro-toxicity, neuro-degeneration and neuro-inflammation studies will be discussed.

In this webinar Susana discusses:

• The basics of live-cell analysis and applications in neuroscience discovery research
• How to gain greater biological insight and productivity with advanced cell models
• Relevant examples for neuro-toxicity, neuro-degeneration and neuro-inflammation 
• Automated assays for neuronal activity, neurite dynamics, cell health and neuro-immune functions (phagocytosis, chemotaxis) in stem cells and primary neuronal cultures

Integrated protocols and live-cell analysis solutions for different 3D tumor models

In the search for greater clinical translation, cancer researchers are increasingly turning to more complex live-cell assay systems that recapitulate key aspects of the tumor microenvironment. These include (1) more relevant tissue organization, (2) bio-matrices containing structural proteins and growth factors, and (3) the presence of stromal and immune cells. In this webinar, we describe integrated protocols and live-cell analysis solutions for different 3D tumor models, ranging from simple mono-culture single spheroid assays through to multi-spheroid tumor/fibroblast and tumor/immune cell co-cultures in a Matrigel biomatrix.

You will learn how to:

• Assemble 96-well 3D spheroid assays in both round-bottom ultra-low attachment microplates, and flat plates on a Matrigel base
• Automatically acquire and analyse images over time, using the new IncuCyte S3 DF Brightfield modality and Spheroid module
• Quantify tumor growth, morphology and viability, either label-free or with fluorescent cell health markers
• Study the impact of stromal cells and immune cells on tumor biology in these 3D systems
• Gain greater biological insight and productivity in your 3D tumor assays

During this webcast Dr. Mayumi Fujita of the National Institutes for Quantum and Radiological Science and Technology in Japan will address the method of real-time imaging of invading cells using the IncuCyte Live-Cell Analysis System, and describe how this model is useful for many studies.

In previous studies, only 1% of cells in the cultured human pancreatic cancer cell line, PANC-1, were capable of invasion through MatrigelÆ in a transwell invasion assay. This suggested that invasive PANC-1 cells have unique characteristics conferring this phenotype. This was further investigated using a 3D spheroid model of PANC-1, embedded in Matrigel, coupled with IncuCyteÆ Live Cell imaging and analysis to capture the movement of the distinct invading population in real time. The identified, invasive PANC-1 were collected and metabolically characterized by CE-TOFMS, and their metabolic profile compared with whole-culture PANC-1. (Fujita et al. 2017, Cancer Science). The invasive PANC-1 cells were distinct from those of the whole cultured PANC-1, with demonstrated increased consumption of ATP, assumed activation of ATP-generating pathways, and higher arginine utilization by NOS. Although they had higher oxidative stress, the invading cells were also more resistant to it with greater survival upon exposure to H2O2. A reduction of intracellular GSH by BSO inhibited PANC-1 invasiveness. Taken together, these results provide a unique metabolic profile for this invasive PANC-1 cell population, as compared to control cells. Such methodology is readily available for similar assessments of invasive cell phenotypes.

You will learn:

• A method will be provided for the real-time imaging of cancer cell invasion from 3D spheroid using the IncuCyteÆ Live-Cell Analysis System.
• The utility of this model to study cancer cell invasion will be discussed, describing methods of invasion, including that by single cells, collected cells, elongated invasion, or rounded invasion.
• Important methods information for real-time image capture, identification of invasive phenotypes, and integration with metabolomics analysis.
• How to easily apply the use of IncuCyte technology and associated applications to their cell line of interest for related studies.

The success of immune checkpoint blockade adds a new therapeutic category to the cancer therapy repertoire. Despite efforts made on cancer cell and immune cell interaction, how cancer cells initiate immune escape is less understood. Here we reveal PD-L1’s immunosuppression activity is stringently modulated by the spatial restraints between ubiquitination and N-linked glycosylation. We identified GSK3beta as a novel PD-L1 interacting protein capable of inducing phosphorylation dependent proteasome degradation. The essence of PD-L1 N192, N200 and N219 glycosylation suggests it antagonizes GSK3beta binding. In this regard, only non-glycosylated PD-L1 forms a complex with GSK3beta and beta-TrCP. More importantly, activation of GSK3beta-mediated PD-L1 degradation by TKI can reduce membrane PD-L1 expression and thereby enhance anti-tumor immunity. To test T cell-mediated cancer cell killing in vitro, we employed a time lapse microscopy, IncuCyte, to monitor RFP-tagged BT549 cells and activated PBMC interaction over a five day period. Massive cancer cell death was observed in glycosylation deficient cancer cells, suggesting glycosylation of PD-L1 is required for its immunosuppression. Together, our results link ubiquitination and glycosylation pathways with stringent regulation of PD-L1, proposing a new therapeutic strategy to enhance cancer immune therapy efficacy.

Learning Objectives:

• Molecular mechanism of PD-L1 regulation in cancer cells
• Immunosuppressive nature of triple negative breast cancer
• Using an in vitro co-culture system to analyze T cell-mediated cancer cell killing effect

Deriving deeper biological insight & improving productivity in immune-cell biology

The most common in vitro methods for immunologists to analyze cells of the immune system are flow cytometry, PCR and various forms of ELISA. Together, these enable quantification of different cell populations at the molecular and functional level (‘immuno-phenotyping’) and measurement of immune responses, for example, cytokine release. While these platforms are extremely powerful, they provide little insight into morphology and the spatial interactions of cells. Moreover, as ‘end-point’ measures, they are not well suited to report on the dynamic changes in biology over time.

In this webinar, Dr. Del Trezise discusses:

• Real-time live-cell analysis
• The use of automated time-lapse imaging for the kinetic quantification of and functional analysis of immune cells
• A number of case studies including T cell proliferation and clustering, phagocytosis, chemotaxis, cytolysis and viral infection assays

Alongside intense efforts to exploit T-cells as immunotherapies for cancer (e.g. checkpoint inhibitors, CAR-T, T-cell metabolism), researchers are increasingly considering other immune cell types for novel targets.  One example is the enhancement of macrophage function via inhibitors of CD47 ìdonít-eat-meî signalling proteins, enabling tumour cells to evade clearance by neighbouring phagocytes (Kim et al, 2012, Leukemia 26, 2538ñ2545).  To identify new CD47 and phagocyte modulators, novel, direct and validated assays with throughput commensurate to drug discovery are required.  

In this mini-webinar Tim Dale will:

• Illustrate how real-time live-cell analysis using pH-sensitive fluorescent probe-labelled target cells can be used to visualize and quantify phagocytosis.
• Share data on the pro-phagocytic effects of anti-CD47 antibodies.
• Demonstrate how other live-cell phenotypic assays (e.g. apoptosis, proliferation) are used to validate mechanisms of action.

Kinetic antibody internalization assays using live-cell analysis

Monoclonal antibodies (mAb) and antibody-drug conjugates (ADCs) are widely used biological therapeutics.  A key property of these biologics is the extent and rate of internalization into different cells, which governs their efficacy, safety and pharmaco-dynamic profile. As a result, quantifying and comparing the rate of Ab internalization of drug candidates is critical in the biopharmaceutical selection and optimization process.

In this mini-webinar Nicola Bevan discusses:

• How real-time live-cell analysis of antibody internalization can provide additional insight.
• The use of the IncuCyte FabFluor-pH reagent to provide a turn-key approach to antibody labelling.
• Examples of how the assay may be used for characterisation of early stage test antibodies and candidates in a biologics discovery paradigm.